ABSTRACT Our over arching goal of this application is to determine if exogenous progestins used in long acting reversible contraceptives (LARCs) will enhance adolescent risk for mucosal HIV acquisition. In the U.S., persons aged 13-24 years accounted for 26% of all new HIV infections. The HIV epidemic in adolescent girls reflects the strong combined impact of gender and income inequality, early sexual debut, age disparate sexual relationships, psychosexual maturation, sociocultural context, and heightened biological vulnerability. Exogenous progestins, expecially medroxyprogesterone acetate (MPA), have been epidemiologically linked to greater HIV acquisition. Gaps remain in our understanding of biological factors that might be associated with increased risk of HIV acqusition in adolescent girls and how exogenous progestins will influence this risk. LARCs use is encouraged for all women desiring to not become pregnant as they release progestins over 3 to 5 years. Typical LARCs include etonogestrel implants (ENG-I) or levonorgestrel-intrauterine system (LNG- IUS). Our preliminary data shows cervical tissue collected from women using MPA and LNG-IUS for contraception replicates HIV 2- to 3.5-fold higher than cervical tissue from non-contracepting women. Therefore, our aims center on potential genital tract differences non-contracepting and contracepting (ENG-I and LNG-IUS) 120 adolescents (18 to 19 years old) and correlate our findings to non-contracepting and contracepting 90 adult women (?25 years old). First, differences in the capcity of cerivcal tissue to replicate HIV will be assess between the groups and associated with resident cell populations. Secondly, the vaginal fluid and epithelium, which provide the first line of defense against pathogens, including HIV, will be systematically studied. Vaginal fluid will be characterized for the microflora and the soluble mucosal proteins using an unbiased proteomic approach. These results will be correlated to functional activity of the luminal fluid as measured by in vitro anti-HIV activity. Our premise is that non-contracepting adolescent women will demonstrate higher levels of mediators and activated immune cells that are associated with increased HIV infection compared to more mature women. However, contracepting adolescent and adult women will have comparable levels of the mediators, activated immune cells, and capacity of tissue to replicate HIV. A key challenge will be the recruitment of adolescent girls. Our advantage is that adolescents frequent the outpatient clinic at Magee Womens Hospital and our clinical team has worked with adolescents in other studies, which overcomes many of the recruitment challenges. We have close working relationships among the groups represented in this application and have optimized specimen handling/processing/testing which is key for reproducibility in our work. This application will uniquely associate quantified analyses with functional outcomes and begin to address if exogenous progestins impose additional risk of HIV acquisition to young women.